Rheumatoid arthritis (RA) is a chronic, inflammatory joint disorder that is considered to be autoimmune, although the autoantigens that trigger and sustain the immune response remain unknown. There are several known genetic and environmental risk factors for development of the disease. The main genetic risk factor is a sequence common to several HLA DRB1 alleles known as the shared epitope (SE) (reviewed in Silman and Pearson (2002) Arthritis Res 4:S265-S272). Environmental factors which affect the risk of RA are thought to include female sex hormones, diet, cigarette smoking and exposure to silica dust (reviewed in Oliver and Silman (2006) Scand J Rheumatol 35: 169-174. Exposure to infection has been suggested to act as a trigger for RA, and a number of agents have been implicated, including Epstein-Barr virus, parvovirus, Proteus and Mycoplasma (Silman and Pearson, supra). Findings which make Epstein-Bar virus a promising candidate include the discovery that antibodies in RA cross-react with EBV nuclear antigen, sequence similarity between EBV glycoprotein and HLA DRB1-SE and EBV DNA load in peripheral blood mononuclear cells of RA patients has been found to be 10 times that of controls (Oliver and Silman, supra; Balandraud, Roudier and Roudier (2004) Autoimmunity Rev 3:362-367). The staphylococcal enterotoxin B has also been suggested to play a critical role in the pathogenesis of RA (Origuchi et al (1995) Ann Rheum Dis 54:713-720), as has human parvovirus B19 (Takahashi et al (1998) Proc Natl Acad Sci USA 95:8227-8232). However, other researchers have cautioned against ignoring the possibility that RA is unrelated to infection, concluding that there is no overwhelming evidence linking any single known infectious agent with RA (Carty, Snowden and Silman (2004) Ann Rheum Dis 63:46-49). According to those authors, reported links could in fact be explained in other ways, for example evidence of the presence of bacteria in synovial fluid could be explained by inflamed synovium acting as a non-specific trap for bacterial fragments. Furthermore, studies identifying elevated antibodies to particular infectious agents were not always reproducible.
Unlike most other autoimmune rheumatic diseases, the dominant autoantigens in RA are unknown. Circulating IgM antibodies that are specific for IgG are present in up to 75% of patients with RA, and are known as rheumatoid factors. However, rheumatoid factors are also present in patients with other diseases and in up to 5% of healthy individuals (Mageed RA: The RF antigen. In Manual of Biological Markers of Disease Edited by: van Venrooij W J, Maini R N. Dordrecht: Kluwer Academic Publishing; 1996:1-27). Other antibodies are also present in sera from patients with RA, including antiperinuclear factor (Nienhuis R L & Mandema E, Ann Rheum Dis 1964, 23:302-305) and antikeratin antibodies (Young B J et al, Br Med J 1979, 2:97-99). Because both antiperinuclear factor and antikeratin antibodies react with human filaggrin (Sebbag M et al, J Clin Invest 1995, 95:2672-2679) they were collectively designated “antifilaggrin antibodies”. It was subsequently reported that binding of anti-filaggrin antibody epitopes is dependent on the presence of citrulline, an amino acid derived from arginine as a result of a post-translational modification catalysed by the enzyme peptidylarginine deiminase (PAD) (Schellekens G A et al, J Clin Invest 1998, 101:273-281; Girbal-Neuhauser E et al, J Immunol 1999, 162:585-594). Anti-filaggrin antibodies have been found at higher concentrations in synovial membrane than in synovial fluid and peripheral blood (Masson-Bessiere C et al, Clin Exp Immunol 2000, 199:544-552) from patients with RA. However, filaggrin is notably absent from the RA joint (Nijenhuis S et al, Clin Chim Acta 2004, 350:17-34). Citrullinated fibrin, on the other hand, is present in interstitial deposits in the synovial membrane (Masson-Bessiere C et al (supra)) and is recognised by anti-filaggrin antibodies. Endogenous citrullination of fibrin has also been demonstrated in murine models of arthritis (Vossenaar E R et al, Arthritis Rheum 2003, 48-2489-2500). These antibodies have collectively been named ACPAs (anti-citrullinated protein antibodies) and also include anti-SA antibodies, which bind citrullinated vimentin (Vossenaar E R et al, Arthritis Res Ther 2004, 6:R142-R150; Vossenaar E R et al, Ann Rheum Dis 2004, 63:373-381; Despres N et al, J Rheumatol 1994, 21:1027-1033). Anti-citrullinated collagen type II antibodies have also been described (Burkhardt et al (2005) Eur. J. Immunol. 35:1643-1652), as well as antibodies recognising a synthetic citrullinated peptide corresponding to a sequence in Epstein-Barr nuclear antigen 1 (EBNA-1) (Anzilotti C et al (2006) J Rheumatol. 33(4):647-51). A further candidate is citrullinated α-enolase (Kinloch et al (2005) Arthritis Research & Therapy 7:R1421-R1429). Sera of RA patients reacted with citrullinated α-enolase, and a commercially available antibody against α-enolase recognized a band co-migrating with purified α-enolase in synovial cell lysates from RA patients. The band which RA sera reacted with was excised and identified by mass spectrometry as citrullinated α-enolase.
The identification of new antigens and epitopes in RA would supplement or complement existing diagnostic and prognostic tests for RA and might also suggest therapeutic strategies.
A single cyclic citrullinated peptide derived from filaggrin (anti-CCP1) has been used as the basis of an ELISA test for rheumatoid arthritis, although the sensitivity of the test was not as high as the sensitivity of IgM rheumatoid factors (reviewed in E. R. Vossenaar W. J. van Venrooij, Clin. Applied Immunol. Rev, 4 (2004) 239-262). A second generation test, anti-CCP2, based on synthetic cyclic citrullinated peptides having no homology with filaggrin or other known proteins is preferred (Vossenaar & van Venrooij (supra)). The anti-CCP2 assay is more sensitive (up to 80%) and specific (97%) for RA than rheumatoid factors are (Nijenhuis S et al, Clin Chim Acta 2004, 350:17-34). Anti-CCPs may occur early in disease (van Gaalen Fa et al, Arthritis Rheum 2004, 50:709-715), or even before clinical manifestations (Rantapaa-Dahlqvist S et al, Arthritis Rheum 2003, 48:2741-2749). Anti-CCP positivity also predicts a more aggressive form of RA (Vencovsky J et al, Ann Rheum Dis 2003, 62:427-430; Forslind K et al, Ann Rheum Dis 2004, 63:1090-1095). Schellekens et al (Arthritis Rheum. (2000) 43(1):155-63) used a cyclic peptide having the sequence HQCHQESTXGRSRGRCGRSGS (SEQ ID NO. 1) where X is citrulline as the basis for a anti-CCP test. WO 01/46222 to Innogenetics N.V. describes synthetic citrulline-containing peptides which are recognised by RA sera. WO 03/050542 and EP 1 456 662 B1 to Stichting voor de Technische Wetenschappen describe a number of peptides that were identified after screening a library of synthetic cyclic citrullinated peptides with a pool of RA sera. The commercial anti-CCP2 assay was developed based on a proprietary composition using some or all of these peptides, to facilitate and standardise the screening of RA sera for the presence of anti-citrullinated protein reactivity. The assay is available from Euro-diagnostica AB, Medeon, SE-205 12 Malmo, Sweden as “Immunoscan RA” (Catalogue No. RA-96RT). Vossenaar & van Venrooij (supra) report that the anti-CCP2 ELISA is commercially available from Euro-Diagnostica, Arnhem, The Netherlands; Axis-Shield, Dundee, Scotland; INOVA, San Diego, USA and that all of these companies use the same type of CCP2 peptides in their assay. The assay has been reported to have a sensitivity of 60%-80%. Average sensitivity is around 70%. However, since anti-CCP positivity is also a severity factor for RA, the sensitivity will depend on the RA-population in which the sensitivity was determined. Although the commercial anti-CCP2 test is useful, it is based on several different artificial synthetic peptides that do not correspond to any known protein sequences, rather than a single peptide derived from a genuine RA epitope. Therefore, reactivity in the anti-CCP2 test may not reflect a causative mechanism. It is also a poor marker of disease subsets. Since the CCP2 test comprises several different epitopes, it diagnoses CCP positive RA, but this group of patients might comprise several disease subsets, with different disease course, that might need different treatments.